Restless legs syndrome (RLS) is a common neurological disorder involving an extreme urge to move the legs accompanied by painful sensations that worsen during periods of rest and sleep. Prevalence of the disorder is higher in females, and increases with age, leading to the hypothesis that RLS is neurodegenerative in nature. The majority of RLS patients suffer from circadian periodic limb movements (PLM), a symptom associated with several neurological abnormalities, which is also more prevalent in females. The pathogenesis of RLS likely involves the loss of spinal cord dopamine (DA) antinociceptive and neuromodulatory motor effects via action at D2/3 receptors, representing a therapeutic target for symptomatic treatment. The overall goal of these studies is to develop a novel mouse model of RLS utilizing neurotoxin-based lesions of A11 diencephalospinal DA neurons and alterations in iron status, combined with measurement of PLM in lesioned animals while awake and during sleep. Selective destruction of A11 neurons projecting to the lumbar spinal cord, while sparing the neurons that project to other levels of the spinal cord should cause deficits in inhibitory regulation of hindlimb movements consistent with symptoms of RLS. Additionally, gender differences in the characteristics of A11 neurons and the effects of iron status will be examined as possible explanations for the higher prevalence of RLS in females. Proposed studies described in three Specific Aims will employ dual label immunohistochemical, stereological, pharmacological, and neurochemical methods to determine the location, distribution, activity, and regulation of A11 diencephalospinal DA neurons projecting to the lumbar spinal cord in male and female mice. Additionally, the effects of selective 1-methyl-4-phenylpyridinium (MPP+) induced lesion of lumbar spinal cord-projecting A11 neurons on DA concentrations in the spinal cord and behavior will be examined. The proposed model has many advantages over currently available lesion-based methods, will enhance the understanding of the roles of gender, iron status, and A11 DA dysfunction in the pathogenesis of RLS, and will utilize a novel behavioral endpoint for the measurement of PLM in mice. Following its development, this novel, reproducible murine model will be used for the screening of DA-modulating pharmacological agents for the development of new treatments for RLS.